The invention relates to methods of producing and packaging cells for cell therapy treatment of neurological disorders.
A large number of human neurological disorders result from the death of neural cells. One strategy for treating a person having such a disorder is to transplant cells (e.g., neurons, glia, precursor cells, progenitor cells, or stem cells) into the patient""s brain. For example, dopamine-secreting cells derived from fetal brain have been used successfully to correct motor deficits induced by acute lesions of the dopaminergic nigrostriatal pathway in rat and monkey animal models for Parkinson""s disease.
A major impediment in the use of cell therapy treatment for the treatment of neurological disorders such as, for example, Parkinson""s disease, stroke, and multiple sclerosis is the lack of an adequate supply of the appropriate cells. In Parkinson""s disease, fetal pig dopaminergic neurons have been tested as a possible solution to the problem posed by the shortage of human cells (Deacon et al., Nature Med. 3:350-353, 1997). Apart from the immunologic problems posed by xenografting, the demonstrated transfer of pig viruses to human cells will likely impede the development of this line of investigation.
Even with the presence of sufficient numbers of cells for transplantation, there is a second fundamental problem that needs to be addressed: the cells, whether they be human or otherwise, and whether they be neurons, precursor cells, or stem cells, need to be properly prepared for transplantation into the brain of the recipient. In the past, approximately 95% of the transplanted cells have failed to survive (Kordower et al., Mov. Disord. 13: 88-95 (Suppl.), 1998). Compounding the problem, current methods of preparing cells for transplantation often reduce cell viability prior to transplantation.
In order to improve cell therapy for the treatment of a patient having a neurological disorder, one must overcome the lack of an adequate supply of cells and improve the viability of the cells prior to and during transplantation. Thus, there is a need for improved culture methods for the production of cells for transplantation, and for improved cell packaging methods for maintaining cell viability during before and during the transplantation procedure.
We have discovered a highly feasible solution to the shortage of cells for cell transplantation therapy by the way of improved in vitro expansion of cells that are the progenitors of neurons and glia. These progenitor cells can be made to differentiate in vitro prior to transplantation, or they can be transplanted as progenitors and allowed to differentiate in vivo. In either case, we prove herewith, novel steps which may be utilized to ensure the viability of these or any other cells following transplantation.
To achieve this goal, we have developed a culture system which results in the expansion in the number of progenitor cells and an increase in the number of dopaminergic neurons. This system can be readily adapted to large scale culture, allowing for the in vitro production of neurons for the treatment of diseases such as Parkinson""s disease.
The culture system of the present invention further results in increased cell viability before and during transplantation. Thus, not only are more cells available for transplantation, but a greater percentage are viable immediately before and following the transplantation. In human cell therapy protocols, this is highly likely to result in greater behavioral improvement in the treated patient.
Accordingly, in a first aspect, the invention features a method for expansion of neural progenitor cells in vitro. The method includes: (a) providing cells that include neural progenitor cells; (b) plating the cells in culture vessels at an average density of 1xc3x97105 to 7xc3x97105 cells/cm2; and (c) culturing the cells in culture medium and under conditions permissible for proliferation of the neural progenitor cells, wherein the volume of medium results in an initial density of between 5xc3x97104 and 1.5xc3x97105 cells per milliliter of medium.
In one preferred embodiment, prior to the plating of the cells, the culture vessels are coated with polyornithine, fibronectin, or a combination thereof.
Preferably, the culture medium includes fibroblast growth factor. In a preferred embodiment, the concentration is between 2 ng and 100 ng per milliliter and, even more preferably, is between 2 ng and 15 ng per milliliter. The culture medium can also include insulin.
The neural progenitor cells can be any neural progenitor cells (e.g., multipotent progenitor cells, precursor cells, or multipotent stem cells) that can differentiate as neurons, glia, or both. The neural progenitor cells can also include cells that contain a transgene. Preferably, the neural progenitors are progenitors of dopaminergic neurons, and are capable of expressing tyrosine hydroxylase and secreting dopamine. In a preferred embodiment, the neural progenitor cells are from a human.
Preferably, the cells are plated as microislands. The microislands, at the time of plating, are, on average, between 2,000 and 25,000 cells per microisland, more preferably, are between 2,000 and 6,000 cells per microisland, and, most preferably, are between 3,000 and 5,000 cells per microisland.
In a second aspect, the invention features a method for preparing cells for transplantation. The method includes: (a) providing a cell suspension that includes single cells, aggregates of fewer than 500 cells, or a combination thereof; and (b) re-aggregating cells so that greater than 50% of reaggregates have between 25 and 500 cells. Preferably, 75% or more of the reaggregates have between 25 and 500 cells and, most preferably, 90% or more of the reaggregates have between 25 and 500 cells. In particularly preferred embodiments, 90% or more of the reaggregates have between 50 and 200 cells or even between 75 and 150 cells.
In one preferred embodiment, the cells in the reaggregate include cells expressing tyrosine hydroxylase. Preferably, at least 20%, 30% or even 50% of the cells are tyrosine hydroxylase-positive. More preferably, the tyrosine hydroxylase-positive cells secrete dopamine. The cells, preferably, are human cells.
In a third aspect, the invention features a cell aggregate for use in transplantation therapy, the aggregate including 25 to 500 cells. Preferably, greater than 20%, 30%, or even 50% of the cells are tyrosine hydroxylase-positive. It is highly desirable that the tyrosine-hydroxylase cells secrete dopamine. In preferred embodiments, the cell aggregate of the third aspect contains 50 to 200 cells or even 75 to 150 cells. While the cells can be from any animal, it is preferred that the aggregates contain human cells.
The present invention provides improved methods for expanding progenitor cell populations, inducing terminal differentiation, and packaging the cells for transplantation.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof and from the claims.